Isoprenoids are an extremely large and diverse group of natural products that have a common biosynthetic origin, a single metabolic precursor, isopentenyl diphosphate (IPP). Isoprenoids includes all substances that are derived biosynthetically from the 5-carbon compound IPP (Spurgeon and Porter, Biosynthesis of Isoprenoid Compounds, pp 3–46, A Wiley-Interscience Publication (1981)). Some isoprenoids are also referred to as “terpenes” or “terpenoids”. Isoprenoids are ubiquitous compounds found in all living organisms. Some of the well-known examples of isoprenoids are steroids (triterpenes), carotenoids (tetraterpenes), and squalene just to name a few.
For many years, it was accepted that IPP was synthesized through the well-known acetate/mevalonate pathway. However, recent studies have demonstrated that this mevalonate-dependent pathway does not operate in all living organisms. An alternate mevalonate-independent for IPP biosynthesis was initially characterized in bacteria and later in green algae and higher plant (Horbach et al., FEMS Microbiol. Lett. 111:135–140 (1993); Rohmer et al., Biochem. 295: 517–524 (1993); Schwender et al., Biochem. 316: 73–80 (1996); Eisenreich et al., Proc. Natl. Acad. Sci. USA 93: 6431–6436 (1996)).
Many steps in the mevalonate-independent isoprenoid pathway are known. For example, the initial steps involve the pyruvate and D-glyceraldehyde 3-Phosphate, to yield 5-carbon compound, D-1-deoxyxylulose-5-phosphate. A gene, dxs, that encodes D-1-deoxyxylulose-5-phosphate synthase (DXS) that catalyzes the synthesis of D-1-deoxyxylulose-5-phosphate was reported in Mycobacterium tuberculosis (Cole et al., Nature, 393:537–544,1998).
Next, the isomerization and reduction of D-1-deoxyxylulose-5-phosphate yields 2-C-methyl-D-erythritol-4-phosphate. One of the enzymes involved in the isomerization and reduction process is D-1-deoxyxylulose-5-phosphate reductoisomerase (DXR). The gene product of dxr that catalyzes the formation of 2-C-methyl-D-erythritol-4-phosphate has been reported in Mycobacterium tuberculosis (Cole et al., supra).
Steps converting 2-C-methyl-D-erythritol-4-phosphate to isopentenyl monophosphate are not well characterized although some steps are known. 2-C-methyl-D-erythritol-4-phosphate is converted into 4-diphosphocytidyl-2C-methyl-D-erythritol in a CTP dependent reaction by the enzyme encoded by the non-annotated gene ygbP. It has been reported that the YgbP protein is present in Mycobacterium tuberculosis, catalyzing the reaction mentioned above (Cole et al., Supra). Recently, ygbP gene was renamed as ispD as a part of isp gene cluster (SwissProt#Q46893) (Cole et al., Supra).
The 2nd position hydroxy group of 4-diphosphocytidyl-2C-methyl-D-erythritol can be phosphorylated in an ATP dependent reaction by the enzyme encoded by ychB gene. The ychB gene product phosphorylates 4-diphosphocytidyl-2C-methyl-D-erythritol resulting in 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate. Cole et al. (Supra) have reported a YchB protein in Mycobacterium tuberculosis. Recently, ychB gene was renamed as ispE as a part of isp gene cluster (SwissProt#P24209) (Cole et al., Supra).
The product of the ygbB gene converts 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate to 2C-methyl-D-erythritol 2,4-cyclodiphosphate. Cole et al. (Supra) reported that ygbB gene product in Mycobacterium tuberculosis (Nature, 393:537–544, 1998). 2C-methyl-D-erythritol 2,4-cyclodiphosphate can be further converted into carotenoids through the carotenoid biosynthesis pathway. Recently, ygbB gene was renamed as ispF as a part of isp gene cluster (SwissProt#P36663). The reaction catalyzed by YgbP enzyme is carried out in CTP dependent manner. Isopentenyl monophosphate and isopentenyl diphosphate (IPP) are formed through a series of reactions not yet characterized but have recently been proposed to be mediated by LytB and GcpE (Cunningham et al., J. Bacteriol., 182:5841–5848, 2000; McAteer et al., J. Bacteriol., 183:7403–7407, 2000).
In E. coli, IPP can be converted to dimethylallyl diphosphate (DMAPP) by an isomerization reaction catalayzed by the idi gene which is dispensible, suggesting that DMAPP and IPP are produced independently (McAteer et al., J. Bacteriol., 183:7403–7407, 2000). There is a broad group of enzymes catalyzing the consecutive condensation of isopentenyl diphosphate (IPP) resulting in the formation of prenyl diphosphates of various chain lengths. Homologous genes of prenyl transferase have highly conserved regions in their amino acid sequences. They are heptaprenyl synthase, geranylgeranyl (C20) diphosphate synthase (Cole et al., Supra), farnesyl (C15) diphosphate synthase which can catalyze the synthesis of five prenyl diphosphates of various lengths.
Formation of C40 phytoene is carried out by crtB gene that encodes phytoene synthase. Phytoene is formed by condensation of two molecules of C20 precursor geranylgeranyl pyrophosphate (GGPP). Phytoene synthase has been isolated from Streptomyces coelicolor (GenBank#T36969).
Further down in the isoprenoid biosynthesis pathway, more genes are involved in synthesis of carotenoid. Pytoene desaturation step is carried out by crtl gene resulting in the formation of lycopene. A gene encoding phytoene dehydrogenase gene, crtl, has been isolated form Streptomyces coelicolor (GenBank#T36968).
Lycopene cyclization is carried out by crtY/L gene product, lycopene cyclase. Lycopene cyclase has been isolated from Deinococcus radiodurans (White et al. Science, 286:1571–1577 (1999)).
Although many genes needed for isoprenoid and carotenoid synthesis synthesis have been characterized, the genes involved in the isoprenoid and/or carotenoid pathways in Rhodococcus bacteria are not described in the existing literature. There are many pigmented Rhodococcus bacteria which suggests that the ability to produce carotenoid pigments is widespread in these bacteria.
The problem to be solved therefore is to isolate the sequences responsible for isoprenoid biosynthesis in Rhodococcus for their eventual use in isoprenoid and carotenoid production. Applicants have solved the stated problem by isolating a nucleic acid fragment from a Rhodococcus erythropolis AN12 strain containing 10 open reading frames (ORFs) encoding enzymes involved in isoprenoid synthesis.